Method of selecting antioxidants for use in topically applied compositions

ABSTRACT

Antioxidant-containing compositions and methods for confirming antioxidant activity of a composition formulated for topical application to skin. Methods for testing a composition for ability to inhibit both ultraviolet radiation-induced lipid peroxidation on skin and ultraviolet radiation-induced reactive oxygen species formation in the stratum corneum. Compositions and methods for treating and preventing photodamage to skin.

This application claims priority to U.S. Provisional Application Nos.61/023,713, filed Jan. 25, 2008, and 61/114,758, filed Nov. 14, 2008.

FIELD OF THE INVENTION

This invention relates generally to compositions applied topically toskin and hair for protection against ultraviolet radiation. Theinvention also relates to methods of selecting antioxidants forinclusion in such compositions.

BACKGROUND OF THE INVENTION

Exposure of skin to ultraviolet radiation (UVR) induces formation offree radicals and oxidants (singlet oxygen, hydroxy radical, hydrogenperoxide, peroxynitrite, superoxide anions, etc.) collectively referredto as reactive oxygen species (ROS) (Hanson K M, Clegg R M.Photochemistry and Photobiology, 2002, 76(1): 57-63; Black H S.Photochem. Photobiol. 1987, 46, 213-221). Formation of UV-induced ROScauses oxidative damage to lipids, proteins and DNA (Vile G F andTyrrell R M. Free Radic. Biol. Med, 1995, 18, 721-722; Chen Q, et al.Proc. Natl. Acad. Sci. USA, 1995, 92, 4337-4341).

Under normal circumstances, low levels of ROS are neutralized by skin'sconstitutive antioxidant defenses. However, research has shown that evensub-erythemal doses of UVR generates such an abundance of ROS thatskin's own antioxidant defenses become overwhelmed, resulting in a buildup of ROS that are free to cause oxidation, which contributes to acute(immunesuppression and photosensitivity disorders) and chronic(photoaging and skin cancer) forms of skin damage (Thiele J J, et al. J.Invest. Dermatol, 1998, 110(5), 756-761; Sander C. S, et al. J. Invest.Dermatol, 2002, 118 (4), 618-625; Thiele J. J: Skin Pharmacol. Appl.Skin Physiol, 2001, 14 (suppl. 1), 87-91; Sander C S, et al.International Soc. Dermatol, 2004, 43, 326-335). EP 1591104 (STADAPharmaceuticals AG) describes the use of antioxidants in pharmaceuticalformulations for protection against infrared radiation.

Antioxidants (Aox) function to neutralize ROS. If the right type andlevel are present within skin where ROS are being formed, Aox should beable to neutralize ROS before they can attack and oxidize otherbiomolecules. Accordingly, it would be useful to have a method todetermine which topical applied antioxidants can be highly effective atneutralizing UVR-induced reactive oxygen species (ROS) within skin.Further, it would be useful to have a method to distinguish compoundswhich may only be effective in solution to scavenge free radicals fromcompounds that may be highly effective antioxidants on skin when exposedto UVR. Further, it would be useful to have a method to determine thecorrect choice and use-level of antioxidants in sunscreen products toprovide extra protection against skin damage caused by UVR-induced ROS.In addition, it would be useful to have a composition that providesprotection from UVR-induced ROS both at the skin surface and deep in theepidermis, for example as far as the basal layer. These and otherobjectives are provided by the invention described herein.

Accordingly the invention described herein provides, inter alia, amethod which comprises two unique ex vivo methods to assess the abilityof topically applied Aox to provide protection against UVR-induced ROSformation within skin's outer layers. The first method uses microscopy,e.g., fluorescence microscopy, to image and quantify ROS formation inthe inner layers of the epidermis, e.g. through to the basal layers, byimaging sections of human skin. The second method quantifies the extentto which the Aox containing composition inhibits peroxidation of lipidsin skin's outer layers. This specification also demonstrates that acommonly used laboratory test to measure efficacy of antioxidants insolution to scavenge free radicals is not predictive of an Aox's abilityto function effectively on more complex biological substrates like skinexposed to UVR. Thus, the present invention provides an advantage overprior art methods to select Aox for use in sunscreen products to ensurethey provide a protective benefit.

All patent and non-patent references cited herein are herebyincorporated in their entirety into this specification by referencethereto. Identification or discussion of any reference in this sectionor any part of this specification shall not be construed as an admissionthat such reference is available as prior art to the presentapplication.

SUMMARY OF THE INVENTION

The present invention provides a method for confirming antioxidantactivity of a composition formulated for topical application to skin,wherein the method comprises testing the composition for ability toinhibit both ultraviolet radiation-induced lipid peroxidation on skinand ultraviolet radiation-induced reactive oxygen species formationthroughout the epidermis.

The invention also provides a method for screening compounds forantioxidant behavior in a composition to be topically applied to skin,wherein the screening method comprises determining the compound'sability to inhibit both ultraviolet radiation-induced skin lipidhydroperoxide formation and ultraviolet radiation-induced reactiveoxygen species formation throughout the epidermis.

This invention also provides a composition for application to skin orhair of a subject, wherein the composition comprises an antioxidantcompound or combination of antioxidant compounds, wherein theantioxidant compound or combination of antioxidant compoundssubstantially inhibit both ultraviolet radiation-induced skin lipidperoxidation and ultraviolet radiation-induced reactive oxygen speciesformation throughout the epidermis.

The invention further provides for a composition for topicalapplication, wherein the composition comprises at least one antioxidantcompound that substantially inhibits ultraviolet radiation-inducedreactive oxygen species formation in the upper layers of the epidermisand at least one antioxidant compound that substantially inhibitsultraviolet radiation-induced reactive oxygen species formation in thelower layers of the epidermis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—shows the extent to which antioxidants (Aox) inhibit UVR-inducedformation of lipid hydroperoxides (LOOH) ex vivo using tape strips tocollect lipids from human skin in the presence of different types andlevels of Aox.

FIGS. 2A through 2I—shows that the addition of antioxidants tosunscreens significantly reduces UV-induced formation of ROS within thestratum corneum. Relative to the control, the SPF 30 sunscreen formulareduced ROS by 39% whereas the SPF 30 formula with 0.5% vitamin E and0.1% Emblica as antioxidants reduced ROS by 73%. The extent of ROSformation is color coded, with blue indicating low and orange or redindicating high ROS levels.

FIGS. 3A through 3I—shows two photon fluorescence microscopy on skinlayers to demonstrate ability of Vitamin E and Emblica antioxidants toreduce UV-induced formation of ROS within the epidermis. A compositioncontaining 0.5% Vitamin E and 0.1% Emblica provide little protectionagainst free radical formation in the lower (basal) layer of theepidermis.

FIGS. 4A through 4J—shows two photon fluorescence microscopy on skinlayers to demonstrate effect of various known antioxidants to reduceUV-induced formation of ROS within the epidermis. Tested antioxidantextracts actually increased rather than decreased free radicals whenexposed to UVR.

FIGS. 5A through 5D—shows two photon fluorescence microscopy on skinlayers to demonstrate effect of various known antioxidants to reduceUV-induced formation of ROS within the epidermis after 1 MED of UVR. Acomposition containing 0.5% Vitamin E and 0.9% Oxynex ST are shown toinhibit formation of ROS within lower epidermal layers.

FIGS. 6A through 6D—shows two photon fluorescence microscopy on skinlayers to demonstrate effect of various known antioxidants to reduceUV-induced formation of ROS within the epidermis after 4 MED of UVR. Acomposition containing 0.5% Vitamin E and 0.9% Oxynex ST are shown toinhibit formation of ROS within lower 30 epidermal layers.

DETAILED DESCRIPTION

The present invention provides a method for confirming antioxidantactivity of a composition formulated for topical application to skin,wherein the method comprises testing the composition for ability toinhibit both ultraviolet radiation-induced lipid peroxidation on skinand ultraviolet radiation-induced reactive oxygen species formationthroughout the epidermis.

The invention also provides a method for screening compounds forantioxidant behavior in a composition to be topically applied to skin,wherein the screening method comprises determining the compound'sability to inhibit both ultraviolet radiation-induced skin lipidhydroperoxide formation and ultraviolet radiation-induced reactiveoxygen species formation throughout the epidermis.

In certain embodiments of the methods of the invention determininginhibition of ultraviolet radiation-induced reactive oxygen speciesformation in the skin comprises imaging skin throughout the epidermisdown to the basal layer using two-photon fluorescence intensity imaging.

In certain embodiments of the method of the invention determininginhibition of UVR-induced skin lipid hydroperoxide formation comprisesdetermining percent lipid hydroperoxide inhibition of the compound incomparison to placebo.

In certain embodiments the methods of the invention comprise the stepsof applying to distinct areas of skin of a subject an antioxidantcontaining composition and a placebo composition to produce anantioxidant skin site containing antioxidant and skin lipids and aplacebo skin site containing placebo and skin lipids; applying a stripto the antioxidant skin site and the placebo skin site to produce anantioxidant strip sample containing antioxidant and skin lipids and aplacebo strip sample containing placebo and skin lipids; removing saidstrip samples from the skin and exposing said strip samples to UVR toform a UVR-induced antioxidant/lipid reaction product on the antioxidantstrip sample and a UVR-induced placebo/lipid reaction product on theplacebo strip sample; separately contacting the antioxidant strip sampleand the placebo strip sample with solvent to prepare a first extractcontaining UVR-induced antioxidant/lipid reaction product and a secondextract containing UVR-induced placebo/lipid reaction product; assayingsaid first and second extracts for lipid hydroperoxide content for eachextract; and comparing the lipid hydroperoxide content of the firstextract to the lipid hydroperoxide content of the second extract.

In certain embodiments the methods of the invention comprise the furthersteps of applying placebo to two distinct sites on the skin of thesubject; producing strip samples from each site; subjecting stripsamples from only one of the two placebo sites to UVR to produce asubset of irradiated placebo strip samples and a subset of nonirradiatedplacebo strip samples; separately contacting nonirradiated placebo stripsamples with solvent to prepare a third extract containing placebo andskin lipids; and assaying said third extract for lipid hydroperoxidecontent to determine background lipid hydroperoxide formation.

In certain embodiments the methods of the invention comprise comparingthe lipid hydroperoxide content of the first extract to the lipidhydroperoxide content of the second extract comprises calculatingpercent lipid hydroperoxide formation by the following formula:

${\% \mspace{11mu} {LF}} = {\frac{\left( {{{LOOH}\; 1} - {{LOOH}\; 3}} \right)}{\left( {{{LOOH}\; 2} - {{LOOH}\; 3}} \right)} \times 100}$

wherein % LF is the percent lipid hydroperoxide formation, LOOH1 is thelipid hydroperoxide content of the first extract, LOOH2 is the lipidhydroperoxide content of the second extract, and LOOH3 is the lipidhydroperoxide content of the third extract.

The art recognizes numerous compounds as having antioxidant properties.As used herein, the term “antioxidant” refers to compounds orcombinations of compounds determined by the methods of the invention tohave a % LF that is less than 100%. As used herein the term “prooxidant”refers to compounds or combinations of compounds that have a % LF thatis greater than 100%. As demonstrated herein, certain compounds referredto in the art as antioxidants actually have a prooxidant behavior whentested according to the methods of the invention, making them unsuitableas ingredients in compositions for topical application, particularly insunscreens, unless present in the combinations as described herein.

The compositions of the invention containing the appropriate Aox cancomprise any form readily known by those of ordinary skill in the art ofpreparing cosmetic compositions. Examples of such include, but are notlimited to, nonionic vesicle dispersions, emulsions, creams, milks,gels, cream gels, ointments, suspensions, dispersions, powders, solids,sticks, foams or sprays. In certainly preferred embodiments, thecomposition can comprise an anhydrous or aqueous solid or paste,emulsion, suspension, or dispersion. Preferable forms of thecompositions include an oil-in-water emulsion, a water-in-oil emulsion,an alcohol solution, or an aerosol formulation.

Thus, the subject invention also provides a cosmetic composition fortopical application to human skin and/or hair, comprising an appropriateAox and amount of Aox determined by the methods described herein.Non-limiting examples of such cosmetic compositions may include suchproducts as moisturizers, cleansers, conditioners, shampoo, body wash,styling gel/lotion, eye cream and eye liner, blush, mascara, foundation,nail polish, polish remover, eye shadow, lipstick, lip gloss, lipliners, lip balms, makeup remover, nail treatment, foot carecompositions, acne treatment, redness/rosacea treatment, varicose/spidervein treatment, anti-aging compositions, sunless tanning compositions,after-sun compositions, concealers, hair color and bleachingcompositions, skin fading/lighteners, body firming lotion, shavingcream, after shave, relaxer, antiperspirants and deodorants, exfoliants,scrubs, liquid hand soap, bubble bath, pain and wound treatmentcompositions, insect repellant, anti-itch and rash cream, styling mousseand foams, perfume, lubricants, body oil, body spray, baby lotion,diaper cream, baby soap, baby shampoo, baby oil, baby wipes, hair-losstreatment, hair spray, depilatory, hair growth inhibitors, hair removalwaxes, personal cleansing, cologne, oil controller, and hand sanitizer.

Examples of antioxidants useful in the compositions of the inventioninclude, but are not limited to, Diethylhexyl syringylidene malonate,Vitamin E, diisopropyl vanillidene malonate (also referred to as DIPVM)and related compounds (described in U.S. Pat. Nos. 6,602,515; 6,831,191;6,936,735; 7,150,876; and 7,166,273), Tetrahydrocurcumenoids, Soybeanzymbiozome fermentum, Red clover extract, Vitis vinifera (grape) seedextract/Brand B, Green tea extract, Pikea robusta extract, Tocopherol(and) vitis vinifera (grape) seed extract, Vitis vinifera (grape) seedextract/Brand A, Phylanthus emblica fruit extract and combinationsthereof. Amounts of antioxidants to be added to the compositions of theinvention are generally between about 0.01% by weight to about 10.0% byweight, preferably between about 0.1% by weight to about 5.0% by weight.Exact amounts can be determined by one of ordinary skill in the artaccording to testing methods described herein.

In certain embodiments the composition of the invention can compriseVitamin E alone as an antioxidant in an amount greater than about 0.05%by weight, in an amount of about 0.1% by weight or greater, in an amountof about 0.25% by weight or greater, and in an amount of about 0.5% byweight or greater. In certain embodiments the composition of theinvention can comprise Vitamin E as an antioxidant and at least oneadditional antioxidant compound.

In certain embodiments the composition of the invention may compriseVitamin E in combination with a pro-oxidant compound as determined bythe methods of the invention, where the presence of Vitamin E in theseembodiments will counteract the pro-oxidant effects of these compoundsto form an antioxidant combination. In certain embodiments of thiscomposition Vitamin E is present in an amount greater than about 0.05%by weight, in an amount of about 0.1% by weight or greater, in an amountof about 0.25% by weight or greater, and in an amount of about 0.5% byweight or greater. Examples of such pro-oxidant compounds that will beuseful in the compositions of the invention that comprise Vitamin Einclude, but are not limited to, Rosemary officinalis oleoresin, RosaGallica extract, Bioactive Photosynthetic complex from green plants,Thermus Thermophillus ferment, ergothiotaine and combinations thereof.Amounts of pro-oxidants to be added to the compositions of the inventionare generally between about 0.01% by weight to about 10.0% by weight,preferably between about 0.1% by weight to about 5.0% by weight. Exactamounts can be determined by one of ordinary skill in the art accordingto testing methods described herein.

In one embodiment the subject invention, the composition can be in theform of an aerosol, wherein the composition is combined with at leastone propellant, which may be any suitable gas that can be compressed orliquefied within a spray dispensing canister and which expands orvolatilizes to vapor or gas form upon exposure to ambient temperatureand pressure conditions to deliver the composition in an aerosol form.Suitable propellants include hydrocarbons having 1 to 5 carbon atoms,including but not limited to methane, ethane, propane, isopropane,butane, isobutane, butene, pentane, isopentane, neopentane, pentene,hydrofluorocarbons (HFCs), chlorofluorocarbons (CFCs), nitrogen, ethersincluding dimethyl ether, and any mixtures thereof. Those of ordinaryskill in the art recognize that in a closed container such as analuminum can or glass bottle, propellants such as dimethyl ethercondense to the liquid state at ambient temperature. Thus, thecomposition in the aerosol container is liquid formulation which cancontain dissolved propellant, undissolved liquid propellant and gaseouspropellant. All of this is under pressure due to the vapor pressure ofthe propellant. In the practice of the subject invention, the propellantcan be present in an amount up to about 90 weight percent, preferablyfrom about 2 weight percent to about 50 weight percent, and morepreferably about 5 weight percent to about 40 weight percent, mostpreferably 30 weight percent, based on the total weight of the aerosolcomposition.

The compositions of the invention can also comprise aerosol foams orso-called mousse compositions. For example, U.S. Pat. No. 6,627,585describes a mousse-forming cleansing shampoo composition comprising afoamable concentrate comprising at least one surfactant, dispersedparticles of a water-insoluble conditioning agent, an aqueous carrier;and an aerosol propellant. U.S. Pat. No. 6,264,964 describes a cosmeticcomposition including a crosslinked non-emulsifying polysiloxaneelastomer and a carboxyvinyl polymer which is in the form of an aerosolfoam in a pressurized system. The propellant may be introduced into themousse composition at the time of filling by using a standard aerosoldispenser, e.g. a spray can arrangement.

The subject invention contemplates the incorporation of Aox withsunscreen actives in sunscreen and sunblock products and any othertopically applied composition where the addition of sunscreen activeagents and/or Aox would not detract from the efficacy of the product noraffect the sunscreening ability of the sunscreen active agents.

The compositions of the present invention may contain a wide range ofadditional, optional components which are referred to herein as“cosmetic components”, but which can also include components generallyknown as pharmaceutically active agents. The CTFA Cosmetic IngredientHandbook, Seventh Edition, 1997 and the Eighth Edition, 2000, which isincorporated by reference herein in its entirety, describes a widevariety of cosmetic and pharmaceutical ingredients commonly used in skincare compositions, which are suitable for use in the compositions of thepresent invention. Examples of these functional classes disclosed inthis reference include: absorbents, abrasives, anticaking agents,antifoaming agents, antioxidants, binders, biological additives,buffering agents, bulking agents, chelating agents, chemical additives,colorants, cosmetic astringents, cosmetic biocides, denaturants, drugastringents, external analgesics, film formers, fragrance components,humectants, opacifying agents, pH adjusters, plasticizers, reducingagents, skin bleaching agents, skin-conditioning agents (emollient,humectants, miscellaneous, and occlusive), skin protectants, solvents,foam boosters, hydrotropes, solubilizing agents, suspending agents(nonsurfactant), sunscreen agents, ultraviolet light absorbers, SPFboosters, waterproofing agents, and viscosity increasing agents (aqueousand nonaqueous).

In the practice of the invention, the composition may contain one ormore sunscreen active agents. For purposes of the present invention, a“sunscreen active agent” or “sunscreen active” shall include all ofthose materials, singly or in combination, that are regarded asacceptable for use as active sunscreening ingredients based on theirability to absorb UV radiation. Such compounds are generally describedas being UV-A, UV-B, or UV-A/UV-B active agents. Approval by aregulatory agency is generally required for inclusion of active agentsin formulations intended for human use. Those active agents which havebeen or are currently approved for sunscreen use in the United Statesinclude organic and inorganic substances including, without limitation,para aminobenzoic acid, avobenzone, cinoxate, dioxybenzone, homosalate,menthyl anthranilate, octyl salicylate, oxybenzone, padimate O,phenylbenzimidazole sulfonic acid, sulisobenzone, trolamine salicylate,titanium dioxide, zinc oxide, diethanolamine methoxycinnamate, digalloytrioleate, ethyl dihydroxypropyl PABA, glyceryl aminobenzoate, lawsonewith dihydroxyacetone, red petrolatum. Examples of additional sunscreenactives that have not yet been approved in the US but are allowed informulations sold outside of the US include ethylhexyl triazone, dioctylbutamido triazone, benzylidene malonate polysiloxane, terephthalylidenedicamphor sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate,diethylamino hydroxybenzoyl hexyl benzoate, bis diethylaminohydroxybenzoyl benzoate, bis benzoxazoylphenyl ethylhexylimino triazine,drometrizole trisiloxane, methylene bis-benzotriazolyltetramethylbutylphenol, and bis-ethylhexyloxyphenolmethoxyphenyltriazine, 4-methylbenzylidenecamphor, and isopentyl4-methoxycinnamate. However, as the list of approved sunscreens iscurrently expanding, those of ordinary skill will recognize that theinvention is not limited to sunscreen active agents currently approvedfor human use but is readily applicable to those that may be allowed inthe future.

In one embodiment of the invention the additional sunscreen active agentcomprises a photoprotecting effective amount of particulates of at leastone inorganic pigment or nanopigment, non-limiting examples of whichinclude titanium dioxide, zinc oxide, iron oxide, zirconium oxide,cerium oxide, or mixture thereof.

The compositions of the invention may also include materials that alsoincrease the SPF of the final composition by such mechanisms as UVradiation scattering and dispersion. Such materials are referred toherein as “UV-radiation scattering agents” and comprise materials thatexhibit UV absorbing activity or exhibit no UV absorbing activity. Anexample of such UV-radiation scattering agents include polymericmaterials, such as the product known as SunSpheres™ (Rohm and Haas;Philadelphia, Pa.) which are described by their manufacturer as hollowstyrene/acrylates copolymer spheres manufactured by emulsionpolymerization. The polymer spheres are said to raise SPF values acrossthe UVA and UVB region by dispersing and/or scattering the incident UVradiation throughout the film of sunscreen present on a surface, such ashuman skin. It is understood that the spheres cause less UV radiation topenetrate into the skin by redirecting the radiation towards theUV-absorbing sunscreen actives in the sunscreen formulation, where theradiation reacts with the sunscreen active molecules and the energy isdissipated as heat. As used herein, the terms “spheres” or “scatteringagents” are not limited by chemical makeup or shape, but comprise anyagent that produces the effect of lengthening the path of incident UVradiation, increasing the statistical likelihood that the radiation willcontact a sunscreen active molecule, i.e., a UV absorbing active agent.These materials may also include UV absorbing materials that alsoexhibit scattering properties such as ZnO (examples include Z-Cote™products available from BASF), TiO₂ (examples include the Solaveil™products available from Uniqema (New Castle, Del., USA)), compounds suchas methylene bis-benzotriazolyl tetramethylbutylphenol, (“Tinasorb™ M”available from Ciba Specialty Chemicals, Inc. (Basel, Switzerland). UVradiation scattering agents are typically present in the formulation inamounts up to about 10% by weight, preferably in ranges of about 0.5% toabout 7.0% by weight, in particularly preferred ranges of 3% to about 5%by weight.

As used herein, the terms “sunless-tanning agent” or “self-tanningcompositions” refer to compositions which, when applied to human skin,impart thereto an appearance similar to that achieved by exposing theskin to natural or artificial sunlight. Examples of sunless tanningactive agents are described in U.S. Pat. Nos. 6,482,397, 6,261,541, and6,231,837. Such sunless tanning compositions typically comprise, inaddition to an artificial tanning effective amount of a self tanningagent, effective amounts of a composition coloring agent and acosmetically acceptable carrier adapted for topical application to humanskin. The self tanning agents can also include those compositionsgenerally accepted in the art for application to human skin, and which,when so applied, react therein with amino acids so as to form pigmentedproducts. Such reactions give the skin a brown appearance similar to thecolor obtained upon exposing it to sunlight for periods of timesufficient to tan the skin. Suitable self tanning agents include,without limitation, alpha-hydroxy aldehydes and ketones, glyceraldehydeand related alcohol aldehydes, various indoles, imidazoles andderivatives thereof, and various approved pigmentation agents. Presentlypreferred herein as self tanning agents are the alpha-hydroxy aldehydesand ketones. Most preferably, the self tanning agent is dihydroxyacetone(“DHA”). Other suitable self tanning agents include, without limitation,methyl glyoxal, glycerol aldehyde, erythrulose, alloxan,2,3-dihydroxysuccindialdehyde, 2,3-dimethoxysuccindialdehyde,2-amino-3-hydroxy-succindialdehyde and2-benzylamino-3-hydroxysuccindialdehyde.

Suitable emulsifiers or surfactants include pharmaceutically acceptable,non-toxic, non-ionic, anionic and cationic surfactants. Examples ofsuitable non-ionic surfactants include glycerol fatty acid esters suchas glycerol monostearate, glycol fatty acid esters such as propyleneglycol monostearate, polyhydric alcohol fatty acid esters such aspolyethylene glycol (400) monooleate, polyoxyethylene fatty acid esterssuch as polyoxyethylene (40) stearate, polyoxyethylene fatty alcoholethers such as polyoxyethylene (20) stearyl ether, polyoxyethylenesorbitan fatty acid esters such as polyoxyethylene sorbitanmonostearate, sorbitan esters such as sorbitan monostearate, alkylglycosides such as cetearyl glucoside, fatty acid ethanolamides andtheir derivatives such as the diethanolamide of stearic acid, and thelike. Examples of suitable anionic surfactants are soaps includingalkali soaps, such as sodium, potassium and ammonium salts of aliphaticcarboxylic acids, usually fatty acids, such as sodium stearate. Organicamine soaps include organic amine salts of aliphatic carboxylic acids,usually fatty acids, such as triethanolamine stearate. Metallic soapsinclude salts of polyvalent metals and aliphatic carboxylic acids,usually fatty acids, such as aluminium stearate. Other classes ofsuitable anionic surfactants include sulfated fatty acid alcohols suchas sodium lauryl sulfate, sulfated oils such as the sulfuric ester ofricinoleic acid disodium salt, and sulfonated compounds such as alkylsultonates including sodium cetane sulfonate, amide sulfonates such assodium N-methyl-N-oleyl laurate, sulfonated dibasic acid esters such assodium dioctyl sulfosuccinate, alkyl aryl sulfonates such as sodiumdodecylbenzene sulfonate, alkyl naphthalene sulfonates such a sodiumisopropyl naphthalene sulfonate, petroleum sulfonate such as arylnapthalene with alkyl substitutes. Examples of suitable cationicsurfactants include amine salts such as octadecyl ammonium chloride,quaternary ammonium compounds such as benzalkonium chloride.

An emollient is an oleaginous or oily substance which helps to smoothand soften the skin, and may also reduce its roughness, cracking orirritation. Typical suitable emollients include mineral oil having aviscosity in the range of 50 to 500 centipoise (cps), lanolin oil,coconut oil, cocoa butter, olive oil, almond oil, macadamia nut oil,aloe extracts such as aloe vera lipoquinone, synthetic jojoba oils,natural sonora jojoba oils, safflower oil, corn oil, liquid lanolin,cottonseed oil and peanut oil. Preferably, the emollient is acocoglyceride, which is a mixture of mono, di- and triglycerides ofcocoa oil, sold under the trade name of Myritol 331 from Henkel KGaA, orDicaprylyl Ether available under the trade name Cetiol OE from HenkelKGaA or a C₁₂-C₁₅ Alkyl Benzoate sold under the trade name Finsolv TNfrom Finetex. One or more emollients may be present ranging in amountsfrom about 1 percent to about 10 percent by weight, preferably about 5percent by weight. Another suitable emollient is DC 200 Fluid 350, asilicone fluid, available Dow Corning Corp.

Other suitable emollients include squalane, castor oil, polybutene,sweet almond oil, avocado oil, calophyllum oil, ricin oil, vitamin Eacetate, olive oil, silicone oils such as dimethylopolysiloxane andcyclomethicone, linolenic alcohol, oleyl alcohol, the oil of cerealgerms such as the oil of wheat germ, isopropyl palmitate, octylpalmitate, isopropyl myristate, hexadecyl stearate, butyl stearate,decyl oleate, acetyl glycerides, the octanoates and benzoates of(C₁₂-C₁₅) alcohols, the octanoates and decanoates of alcohols andpolyalcohols such as those of glycol and glyceryl, ricinoleates esterssuch as isopropyl adipate, hexyl laurate and octyl dodecanoate,dicaprylyl maleate, hydrogenated vegetable oil, phenyltrimethicone,jojoba oil and aloe vera extract.

Other suitable emollients which are solids or semi-solids at ambienttemperatures may be used. Such solid or semi-solid cosmetic emollientsinclude glyceryl dilaurate, hydrogenated lanolin, hydroxylated lanolin,acetylated lanolin, petrolatum, isopropyl lanolate, butyl myristate,cetyl myristate, myristyl myristate, myristyl lactate, cetyl alcohol,isostearyl alcohol and isocetyl lanolate. One or more emollients canoptionally be included in the formulation.

A humectant is a moistening agent that promotes retention of water dueto its hygroscopic properties. Suitable humectants include glycerin,polymeric glycols such as polyethylene glycol and polypropylene glycol,mannitol and sorbitol. Preferably, the humectant is Sorbitol, 70% USP orpolyethylene glycol 400, NF. One or more humectants can optionally beincluded in the formulation in amounts from about 1 percent to about 10percent by weight, preferably about 5 percent by weight.

A dry-feel modifier is an agent which when added to an emulsion, impartsa “dry feel” to the skin when the emulsion dries. Dry feel modifiers caninclude talc, kaolin, chalk, zinc oxide, silicone fluids, inorganicsalts such as barium sulfate, surface treated silica, precipitatedsilica, fumed silica such as an Aerosil available from Degussa Inc. ofNew York, N.Y. U.S.A. Another dry feel modifier is an epichlorohydrincross-linked glyceryl starch of the type that is disclosed in U.S. Pat.No. 6,488,916.

It may be advantageous to incorporate additional thickening agents, suchas, for instance, various Carbopols available from Noveon Co.Particularly preferred are those agents which would not disrupt thelamellar structure in the formulation of the final product, such asnon-ionic thickening agents. The selection of additional thickeningagents is well within the skill of one in the art.

Additional natural or synthetic substances can also added to thecompositions of the invention to protect from or delay its deteriorationdue to the action of oxygen in the air (oxidation). They may also reduceoxidation reactions in skin tissue. Such substances prevent oxidativedeterioration which may lead to the generation of rancidity andnonenyzymatic browning reaction products. Typical suitable substancesinclude propyl, octyl and dodecyl esters of gallic acid, butylatedhydroxyanisole (BHA, usually purchased as a mixture of ortho and metaisomers), butylated hydroxytoluene (BHT), green tea extract, uric acid,cysteine, pyruvate, nordihydroguaiaretic acid, Vitamin A, Vitamin E andVitamin C and their derivatives. One or more such substances canoptionally be included in the composition in an amount ranging fromabout 0.001 to about 5 weight percent, preferably about 0.01 to about0.5 percent.

Chelating agents are substances used to chelate or bind metallic ions,such as with a heterocylic ring structure so that the ion is held bychemical bonds from each of the participating rings. Suitable chelatingagents include ethylene diaminetetraacetic acid (EDTA), EDTA disodium,calcium disodium edetate, EDTA trisodium, albumin, transferrin,desferoxamine, desferal, desferoxamine mesylate, EDTA tetrasodium andEDTA dipotassium, or combinations of any of these.

Fragrances are aromatic substances which can impart an aestheticallypleasing aroma to the sunscreen composition. Typical fragrances includearomatic materials extracted from botanical sources (i.e., rose petals,gardenia blossoms, jasmine flowers, etc.) which can be used alone or inany combination to create essential oils. Alternatively, alcoholicextracts may be prepared for compounding fragrances. However, due to therelatively high costs of obtaining fragrances from natural substances,the modern trend is to use synthetically prepared fragrances,particularly in high-volume products. One or more fragrances canoptionally be included in the sunscreen composition in an amount rangingfrom about 0.001 to about 5 weight percent, preferably about 0.01 toabout 0.5 percent by weight. Additional preservatives may also be usedif desired and include well known preservative compositions such asbenzyl alcohol, phenyl ethyl alcohol and benzoic acid, diazolydinyl,urea, chlorphenesin, iodopropynyl and butyl carbamate, among others.

The compositions of the invention can further comprise skin protectantactive agents. Suitable examples include (with preferred weight percentranges), Allantoin (0.5 to 2 percent); Aluminum hydroxide gel (0.15 to 5percent); Calamine (1 to 25 percent); Cocoa butter (greater than 50);Cod liver oil (5 to 14 percent); Colloidal oatmeal; Dimethicone (1 to 30percent); Glycerin (20 to 45 percent); Hard fat (greater than 50);Kaolin (4 to 20 percent); Lanolin (12.5 to 50 percent); Mineral oil(greater than 50 percent); Petrolatum (greater than 30 percent); Sodiumbicarbonate; Topical starch (10 to 98 percent); White petrolatum(greater than 30 percent); Zinc acetate (0.1 to 2 percent); Zinccarbonate (0.2 to 2 percent); and Zinc oxide (1 to 25 percent).

The compositions of the invention may further include insect repellingcomponents. The most widely used insect repelling active agent forpersonal care products is N,N-Diethyl-m-toluamide, frequently called“DEET” and available in the form of a concentrate containing at leastabout 95 percent DEET. Other synthetic chemical repellents include ethylbutylacetylaminoproprionate (also known as IR 3535), dimethyl phthalate,ethyl hexanediol, indalone, di-n-propylisocinchoronate, bicycloheptene,dicarboximide and tetrahydrofuraldehyde. Certain plant-derived materialsalso have insect repellent activity, including citronella oil and othersources of citronella (including lemon grass oil), limonene, rosemaryoil and eucalyptus oil. Choice of an insect repellent for incorporationinto the sunscreen emulsion will frequently be influenced by the odor ofthe repellent. The amount of repellent agent used will depend upon thechoice of agent; DEET is useful at high concentrations, such as up toabout 15 percent or more, while some of the plant-derived substances aretypically used in much lower amounts, such as 0.1 percent or less.

Topical application of the compositions of the invention describedherein to the hair or skin of a human will provide enhanced protectionagainst deleterious effects of ultraviolet radiation (UVR). Thus, thesubject invention further provides a method for protecting human skinand/or hair against the deleterious effects of solar radiation, moreparticularly UVR, which method comprises topically applying thereto aneffective amount of the compositions as described herein containingsunscreens and one or more antioxidants. An esthetically beneficialresult of exposure of skin to UVR (i.e., light radition wavelengths offrom 280 nm to 400 nm) is the promotion of tanning of the humanepidermis. Another benefit of sun exposure comes from production ofvitamin D within the skin. UVR is typically divided into UV-A (lightwavelengths from 320 to 400 nm) and UV-B (wavelengths ranging from 280to 320 nm) regions. Overexposure to UV-B irradiation is generallyunderstood to lead to skin burns and erythema. In addition, overexposureto UV-A radiation may cause a loss of elasticity of the skin and theappearance of wrinkles, promoting premature skin aging. Such irradiationpromotes triggering of the erythemal reaction or amplifies this reactionin certain individuals and may even be the source of phototoxic orphotoallergic reactions. It is increasingly believed that overexposureto UV-A may also lead to melanoma. Thus, the application of thecompositions of the invention to the skin and/or hair of an individualwill provide enhanced UVR photoprotection (UV-A and/or UV-B) of the skinand/or hair of the individual.

The invention further provides a method of treating and/or reversingphotodamage of skin by applying the compositions of the invention toskin that will be or has been exposed to UVR. The term “treating and/orreversing photodamage” is intended to mean obtaining an improvement inone or more attributes of skin condition such as dryness, texture,elasticity/firmness/resiliency, lines/wrinkles, skin tone/clarity,uniformity of pigmentation, and/or erythema which condition isexacerbated by exposure to UVR.

The sunscreen containing compositions of the invention are intended toprovide a sun protection factor (SPF) rating of at least 2, withadditional preferable embodiments having a sun protection factor of atleast 5, at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 55, atleast 60, at least 65, at least 70, at least 75, at least 80, at least85, at least 90, at least 95, and at least 100. The sunscreen containingcompositions of the invention are also intended to provide U.S. FDA UV-B“star ratings” of at least one star, at least two stars, at least threestars and up to four stars.

The invention will be further described by means of the followingexamples, which are not intended to limit the invention, as defined bythe appended claims, in any manner.

EXPERIMENTAL DPPH (α,α-diphenyl-β-picrylhydrazyl) free radical test

DPPH is a stable free radical that when dissolved in solution forms anintense purple color. When reduced by an antioxidant, the purple colorfades until it finally disappears as DPPH is completely reduced. Theextent to which the color fades can be easily measured and used to rankthe relative effectiveness of different materials purported to haveantioxidant properties.

Measurements were recorded for antioxidant raw materials in simplemethanol solutions. Samples were prepared by dissolving antioxidants atvarious concentrations in methanol. After sample preparation, 125 μl ofthe sample or pure methanol as a control were pipetted into sample testtubes followed by 2 ml of methanol and vortexed. Then 2 ml of DPPH stocksolution (0.25 mM in methanol) was added to each tube (giving a totalvolume of 4.125 ml for each sample) and vortexed. Immediately afteraddition of DPPH, test tubes were covered and placed into a 30° C. waterbath for 20 minutes. After the 20 minute incubation, the absorbance ofeach sample was recorded at 517 nm using a Perkin Elmer Lambda 40spectrophotometer. All samples were prepared in triplicate and theirmean absorbance values were used to express the efficacy of antioxidantsat various concentrations in terms of antioxidant reducing units (ARU)by using the following equation:

ARU=(Absorbance Methanol Control−Absorbance sample)×10

ARU values range in magnitude from 0 for “no” efficacy to about 15 forraw materials that have high antioxidant efficacy.

Antioxidant effectiveness for a variety of oil and water-soluble rawmaterials purported to have antioxidant properties appears in Table 1.The raw materials include well-known antioxidants such as vitamin E, inaddition to popular plant extracts such as green tea, rose, grape andmushrooms, among others. Effectiveness is expressed as antioxidantreducing units (ARU), which span values of 0 for no efficacy to 12 forantioxidants with high efficacy to reduce the DPPH radical. Examples ofboth oil and water soluble antioxidants spanned this range.

TABLE 1 Antioxidant Level (%) ARU Solubility Rosemary officinalisoleoresin [ROO] * 0.1 0.0 O Arjunolic acid (1%) 1.0 0.4 O Diethylhexylsyringylidene malonate 1.0 5.7 O Vitamin E [Vit. E] * 0.5 6.5 OTetrahydrocurcumenoids [THC]* 0.5 9.0 O Bioactive photosynthetic complex0.1 0.0 W from green plants [BPSC]* Thermus thermophillus ferment[Thermus]* 1.0 0.0 W Ergothiotaine [ET]* 0.1 0.0 W Rosa gallica extract[Rosa G]* 1.0 0.0 W Foeniculum vulgare (fennel) seed Extract 5.0 0.0 WSoybean zymbiozome fermentum 1.0 0.0 W Shitake mushroom extract 5.0 0.0W Helianthus annuus (sunflowler) extract 1.0 0.0 W Red clover extract1.0 0.6 W Vitis vinifera (grape) seed extract/Brand B 0.1 1.9 W Greentea extract [GT]* 0.1 2.2 W Pikea robusta extract 1.0 2.2 W Tocopherol(and) vitis vinifera 1.0 7.6 W (grape) seed extract n-Acetyl cysteine[NAC] * 0.1 9.9 W Vitis vinifera (grape) seed extract/Brand A 0.1 11.5 WPhylanthus emblica fruit extract [Emblica]* 0.1 11.5 W

Compared with vitamin E, some materials clearly possess lower (ARU<6.5)while others higher (ARU>6.5) antioxidant effectiveness. Based upon ARUvalues alone, it would be expected that antioxidants with ARU>6.5 wouldbe superior to vitamin E in their ability to neutralize UVR-inducedformation of ROS within the skin. However, recognizing that ROS covers awide range of reactive compounds, including free radicals but also otheroxidants like hydrogen peroxide, singlet oxygen or peroxynitrite,results from the DPPH free radical test may not adequately predictantioxidant effectiveness on skin that is exposed to UVR. We, therefore,selected several antioxidants (denoted with *) from Table 1 covering arange of ARU values to test in model systems that more closely mimicintact human skin exposed to UVR to understand if Aox effectiveness insolution as measured by the DPPH free radical test translated to similarlevels of effectiveness on skin.

Ex Vivo Tape Strip Method to Assess Antioxidants Ability to InhibitLipid Peroxidation.

To determine if antioxidants (Aox) maintain their effectiveness on skinin the presence of UVR, we devised a novel and more relevant model thatutilizes human skin lipids as substrates for UVR-induced peroxidation.Lipids removed from skin on broad pieces of tape serve as the substratesfor subsequent exposure to UVR. By applying a standard lotion with orwithout Aox to skin prior to tape-stripping, skin lipids can becollected on tape strips in the presence or absence of Aox thatessentially maintains the same proximity that lipids and Aox had onskin. Following UVR exposure, the extent to which the presence of Aoxprotect lipids against peroxidation from ROS can be measured relative tolipids in the absence of antioxidants.

Human volunteers were recruited for the test and asked not to apply anyproducts to their arms for at least two days before the test Prior toany product treatments, inner aspects of subjects' left and rightforearms were wiped with a Kimwipe™ moistened with isopropanol to removeany residues that might be on the surface of the skin. Arms were wipedonly once applying gentle pressure and then allowed to dry at least 10minutes before proceeding. A template (90 mm×50 mm) was positioned oneach inner forearm such that two areas could be clearly delineated.Using a superfine tip Sharpie™ pen, a mark was placed at each corner ofthe template to outline each application site, with two sites delineatedper forearm. Using a fingercot, either placebo or antioxidant lotion wasapplied (100 mg) to a delineated site on a forearm. Care was taken toinsure that products were applied evenly within the entire applicationarea. After application, sites were allowed to air-dry for 30 minutesduring which subjects were instructed not to allow any clothing to comeinto contact with test areas.

After lotions dried for 30 minutes, each site was tape-stripped using a4.0 inch piece of Scotch® Brand No. 800 Prescription Label tape (1.5inches wide). One end of the tape was folded over to provide an edgethat did not adhere to skin for easy removal. The piece of tape waspositioned over a site and then using a finger the tape was gentlypressed onto the skin to make good contact. Then the tape was quicklyremoved from the subject's arm. After removal, all tapes were stored ina dark location such as a drawer until they were either irradiated withUV or extracted with isopropanol (i.e., nonirradiated control). Selecttape strips of skin of each subject were irradiated with a dose of 10joules/cm² using a 1000 W Xe arc solar simulator (WG320 filtered). AnOptronics OL-754 spectroradiometer was used to adjust the output of thesolar simulator to deliver a constant dose of UVR. After irradiation,tapes were trimmed to a length of three inches and placed in 20 ml glassscintillation vials. Then four ml of isopropanol was added to each vial,after which they were capped. Vials were then shaken vigorously againand placed in a −20° C. freezer to extract overnight. The next daysamples were shaken before aliquots were removed for lipid hydroperoxide(LOOH) analysis.

Each tape extract was assayed for total LOOH content using a LipidHydroperoxide Assay Kit (Kamiya Biomedical Company, Thousand Oaks,Calif.) following manufacturer's directions. Lipid hydroperoxides werethen quantitated by measuring methylene blue formation at 675 nm using aspectrophotometer. Standard curves were prepared using cumenehydroperoxide and were linear over the range of LOOH detected in theseexperiments. Each extract was assayed in triplicate and the resultspresented here represent the mean of those analyses. The standarddeviations were typically less than 10%.

The extent to which antioxidant (Aox) or placebo lotions inhibitedUVR-induced lipid hydroperoxide (LOOH) formation was calculated byinserting the values of LOOH determined from the four application siteson each volunteer into the following equation:

${\% \mspace{14mu} {LOOH}\mspace{14mu} {Formation}} = {\frac{\left( {{{LOOH}\mspace{14mu} {Aox}\mspace{14mu} {irradiated}} - {{LOOH}\mspace{14mu} {unirradiated}\mspace{14mu} {placebo}}} \right)}{\left( {{{LOOH}\mspace{14mu} {irradiated}\mspace{14mu} {placebo}} - {{LOOH}\mspace{14mu} {unirradiated}\mspace{14mu} {placebo}}} \right.} \times 100}$

Calculation of “% LOOH formation” enables each subject to be his owninternal control and normalizes the data with respect to the area of thetape (3 inches) used to strip skin. In this way, values for % LOOHformation can be compared between sites on different people.

Data were analyzed using paired t-tests to determine if antioxidant orplacebo treatments yielded significantly different results. An alphalevel of 0.05 and a power of 80% was used for all statistical tests.

Results for select antioxidants (* in Table 1) to protect lipids fromUVR-induced ROS formation appear in FIG. 1, which reveals severalstriking features. Both water and oil soluble materials can protectlipids against UVR-induced peroxidation; however, the raw material mustbe able to partition into the lipid bilayers to be protective. Vitamin Eprotects skin lipids in a dose dependant manner (yellow bars).Tetrahydrocurcurminoids (THC) are as efficacious as Vitamin E. Vitamin Ein combination with Emblica, GT or THC (orange bars) protect about aswell as Vit. E alone (yellow bars). Surprisingly, some materials (redbars) increase rather than decrease lipid hydroperoxide (LOOH) levels,acting as pro-oxidants as opposed to antioxidants. Addition of Vitamin Ecan reduce pro-oxidant properties (blue bars) of antioxidants but not tothe same degree as observed with Vitamin E alone.

These results demonstrate that the DPPH free radical test by itself isnot predictive of an Aox's ability to function effectively on skin toprotect lipids from peroxidation from UV-induced ROS formation.

Two-Photon Fluorescence Microscopy Imaging of Stratum Corneum.

Two-photon fluorescence intensity imaging was performed ex vivo onpieces of human breast skin (˜0.5 cm×0.5 cm) to detect and quantifylevels of UV-induced reactive oxygen species (ROS) in the stratumcorneum using procedures described previously (Hanson K M, Clegg R M.Photochemistry and Photobiology, 2002, 76(1): 57-63). Test formulas wereapplied to the surface of skin samples at 2 mg/cm² using the tip of aglass rod. Prior to irradiation, skin samples were incubated in 100μmolar dihydrorhodamine (DHR) in phosphate-buffered saline-ethanol. DHRpartitions into the tissue where it reacts with UV-induced ROS toproduce highly fluorescent rhodamine-123, which is subsequently imagedand quantified as a measure of UV-induced ROS formation. At least twounique areas are imaged from each skin sample and at each depth. A baseformula without sunscreen actives or antioxidants was used as a control

The images shown in FIG. 2 demonstrate the extent of UV-induced ROSformation that resulted within the full thickness of stratum corneumafter each formula was applied to intact pieces of human skin andirradiated. The images demonstrate clearly that exposure to 4 MEDs offull spectrum UV radiation generates abundant ROS formation. Applicationof an SPF 30 broad spectrum sunscreen formula prior to irradiationreduced ROS formation by 39% relative to the control formula by virtueof its ability to absorb UV before it can interact with skin to generateROS. However, application of an SPF 30 formula plus antioxidants (0.5%vitamin E, 0.1% Emblica) reduced ROS formation by a total of 73%relative to the control formula, which represents an additionalreduction in ROS of 34% compared with the formula that only contains thesunscreen actives.

Thus, these results demonstrate convincingly that addition ofantioxidants of the right type and level can complement sunscreens as anadditional strategy to protect skin from the harmful effects ofUV-induced ROS formation. Exposure of skin to UVR can generate anabundance of ROS even through a protective layer of broad spectrum SPF30 sunscreen. With the power to neutralize ROS, antioxidants (Aox) canprovide measurable and meaningful levels of protection against thedamaging effects of ROS and in this way significantly augment theprotective power of sunscreens provided, however, that Aox for use insunscreens are selected appropriately.

Protection Through Basal Layer with Antioxidant Combination

A. Lipid Peroxidation Inhibition

Vitamin E is highly effective at neutralizing UV-induced ROS in theouter layer of epidermis, the stratum corneum. Placebo and experimentalformulations were prepared to compare the formulations' ability toinhibit lipid peroxidation. The formulations used are shown in Table 2.

TABLE 2 Placebo and Antioxidant Lotions Used in Tape Strip StudiesPlacebo Placebo Antioxidant Antioxidant Lotion Lotion Lotion LotionIngredient %, w/w %, w/w %, w/w %, w/w Part A USP purified 86.75 86.7586.15 85.35 water Simulgel NS 2.00 — 2.00 Sepigel 305 — 2.00 2.00 —Sodium cetearyl 0.250 0.25 0.25 0.25 sulfate Emblica 0.1 — Part B Octylpalmitate 10.0 — 10.0 Isopropyl laurate — 10.0 10.0 — Vitamin E — 0.500.50 Oxynex ST — — 0.90 Part C Germaben II 1.00 1.00 1.00 1.00The formulations were prepared by adding sodium cetearyl sulfate to thewater of part A and mixing, followed by addition of either Simulgel NSor Sepigel 305 and mixing thoroughly. Then the Part B ingredients wereadded with mixing, followed by Germaben II in Part C. After allingredients are added, the emulsion was mixed thoroughly.

Using the ex vivo tape strip method as described above, a compositioncontaining vitamin E, by itself or in combination with Emblica, wasfound to be highly effective at protecting skin's lipids on its outersurface from UV-induced oxidation mediated by ROS (Table 3).

TABLE 3 % Inhibition of Antioxidant LOOH 0.05% vit E −3.40 0.10% vit E28.4 0.25% vit E 61.0 0.1% Emblica 19.6 0.3% Emblica 10.9 0.05% vit E +0.1% 28.1 Emblica 0.10% vit E + 0.1% 47.8 Emblica 0.25% vit E + 0.1%65.0 Emblica 0.50% vit E + 0.1% 74.4 Emblica

B. Two Photon Fluorescence Microscopy

Two photon fluorescence microscopy was then conducted on a formulationcontaining Vitamin E and Emblica to determine ability to inhibitformation of ROS. The methods used were similar to those as describedabove, using confocal microscopy to visualize cells at different depthswithin intact pieces of skin and then fluorescence to quantify theextent of ROS formation. In the present experiment, however, instead ofusing human breast skin, the skin used was the EpiDerm™ Skin Model(MatTek Corporation, Mass. USA), which consists of normal, human-derivedepidermal keratinocytes which have been cultured to form a multilayered,highly differentiated model of the human epidermis. Ultrastructurally,the EpiDerm™ Skin Model closely parallels human skin, thus providing auseful in vitro model to study the ability of antioxidants to neutralizeROS formed during exposure to UVR down to the basal layer.

Prior to UVR exposure, pieces of EpiDerm™ skin were treated withdihydrorhodamine, which partitions throughout the aqueous and lipidregions of the tissue. Upon exposure to UVR, dihydrorhodamine in thetissue reacts chemically with ROS wherever it forms to generate a highlyfluorescent molecule. The fluorescence is subsequently detected andquantified to provide an indication of the level of UV-induced ROSformed in deeper layers of the skin. By applying antioxidants topicallybefore irradiation ROS formation can be measured and compared to the ROSformed after a placebo lotion without antioxidants was applied to theskin. In this way, the efficacy of antioxidants to neutralize ROS withindeeper layers of the skin can be measured and their relativeefficiencies established.

Using this method, the experimental formulation containing vitamin E andemblica was tested to determine its ability to neutralize ROS within thebasal layer or bottom layer of the epidermis after exposure to 4 MED.The experimental formulation and placebo lotion used in this experimentwere prepared as follows.

TABLE 4 Placebo Lotion Part A USP purified water 60.04% Acrylates/c10-30alkyl acrylate crosspolymer  0.30% Part B Propylene glycol  5.00%Disodium EDTA  0.01% Triethanolamine, 99%  0.35% Part C Octyl palmitate29.00% PVP/Eicosene copolymer  2.00% Stearic acid  0.50% Polyglyceryl-3distearate  0.29% Methylparaben  0.30% Sorbitan isostearate  0.71%Propylparaben  0.10% Dimethicone, 50 cst  0.40% Part D Benzyl alcohol 1.00%

TABLE 5 Antioxidant Lotion Part A Usp purified water 58.54%Acrylates/c10-30 alkyl acrylate crosspolymer  0.30% Part B Propyleneglycol  5.00% Disodium EDTA  0.01% Triethanolamine, 99%  0.35% Part COctyl palmitate 29.00% Pvp/eicosene copolymer  2.00% Stearic acid  0.50%Polyglyceryl-3 distearate  0.29% Methylparaben  0.30% Sorbitanisostearate  0.71% Vitamin E, dl alpha tocopherol  0.50% Emblica  0.10%Propylparaben  0.10% Dimethicone, 50 cst  0.40% Part D Benzyl alcohol 1.00%A water phase was created by adding Acrylates/C10-30 Alkyl AcrylateCrosspolymer to water of Part A while stirring and mixed until clear andlump-free. While mixing, the propylene glycol and disodium EDTA wereadded to the water phase mixture of Part A and mixed well for 10minutes. Triethanolamine of Part B was then added to the water phasemixture and continued mixing well. Separately an oil phase was createdby mixing the ingredients of Part C together and heat to 140-145° F.while mixing well. The oil phase was then added to the water phase andcontinued mixing to form an emulsion. The emulsion was cooled to roomtemperature and then benzyl alcohol of part D was added to the coolemulsion and mixed thoroughly. Additional water was added QS to weight.The difference between the placebo and experimental formulation was theaddition of antioxidants into the oil phase.

As shown in FIG. 3, while highly effective in the outer layers of theepidermis, the combination of 0.5% vitamin E and 0.1% emblica onlyreduced ROS formation in the basal layer of epidermis by about 5%.

We next evaluated a wide range of ingredients used within the cosmeticindustry for their claimed antioxidant ability, and were surprised toobserve that many of them behaved as pro-oxidants at the basal layer onskin exposed to UVR. As demonstrated in FIG. 4, rather than decreasinglevels of ROS, these ingredients increased levels of ROS by up to 250%within the basal layer of epidermis relative to a placebo lotion withoutany antioxidant. Many of these ingredients that behaved as pro-oxidantsrepresented natural plant extracts from fennel seeds, rose and whitegrapes. Together these results demonstrated that antioxidants used insuncare products where intentional sun exposure occurs need to beselected judiciously. Moreover the tests demonstrate that the methods ofthe invention reveal that not all ingredients identified as“antioxidants” provide actual antioxidant properties in real world usewith UVR exposures.

We next tested an experimental formulation containing a combination ofantioxidant vitamin E and diethylhexyl syringylidene malonate (Oxynex®ST, Merck KGaA, Germany) to determine whether the combination would beeffective protection against UV-induced ROS formation throughout theepidermis. Placebo and experimental formulations were prepared similarto the methods described above:

TABLE 6 Placebo Lotion Part A USP purified water 60.04% Acrylates/c10-30alkyl acrylate crosspolymer  0.30% Part B Propylene glycol  5.00%Disodium EDTA  0.01% Triethanolamine, 99%  0.35% Part C Octyl palmitate29.00% PVP/Eicosene copolymer  2.00% Stearic acid  0.50% Polyglyceryl-3distearate  0.29% Methylparaben  0.30% Sorbitan isostearate  0.71%Propylparaben  0.10% Dimethicone, 50 cst  0.40% Part D Benzyl alcohol 1.00%

TABLE 7 Antioxidant Lotion Part A Usp purified water 58.54%Acrylates/c10-30 alkyl acrylate crosspolymer  0.30% Part B Propyleneglycol  5.00% Disodium EDTA  0.01% Triethanolamine, 99%  0.35% Part COctyl palmitate 29.00% Pvp/eicosene copolymer  2.00% Stearic acid  0.50%Polyglyceryl-3 distearate  0.29% Methylparaben  0.30% Sorbitanisostearate  0.71% Vitamin E, dl alpha tocopherol  0.50% Diethylhexylsyringylidene malonate  0.90% Propylparaben  0.10% Dimethicone, 50 cst 0.40% Part D Benzyl alcohol  1.00%

A water phase was created by adding Acrylates/C10-30 Alkyl AcrylateCrosspolymer to water of Part A while stirring and mixed until clear andlump-free. While mixing, the propylene glycol and disodium EDTA wereadded to the water phase mixture of Part A and mixed well for 10minutes. Triethanolamine of Part B was then added to the water phasemixture and continued mixing well. Separately an oil phase was createdby mixing the ingredients of Part C together and heat to 140-145° F.while mixing well. The oil phase was then added to the water phase andcontinued mixing to form an emulsion. The emulsion was cooled to roomtemperature and then benzyl alcohol of part D was added to the coolemulsion and mixed thoroughly. Additional water was added QS to weight.The difference between the placebo and experimental formulation was theaddition of antioxidants into the oil phase.

As shown in the Table 8 and FIGS. 5 and 6, in vitro ROS resultsdemonstrate that a lotion containing both Vitamin E and Oxynex® STtogether provided substantially higher protection from UV-induced ROSformation than can be achieved by Vitamin E and Emblica.

TABLE 8 % ROS Neutralized Antioxidant in the Basal Layer After 1 MEDExposure 0.5% vitamin E + 0.1% Emblica ND 0.5% vitamin E + 0.9% Oxynex47 ST After 4 MED Exposure 0.5% vitamin E + 0.1% Emblica 5 0.5% vitaminE + 0.9% Oxynex 33 ST ND = not determined.The addition of Oxynex® ST to a lotion containing Vitamin E neutralized33% ROS after exposure to 4 MED of UVR and neutralized 47% ROS after 1MED UVR within the basal layer. These results are significantly betterthan 5% ROS neutralization for a lotion containing only Vitamin E andEmblica.

The compositions containing the combination of 0.5% Vitamin E and 0.9%Oxynex® ST were also tested in the lipid peroxidation tests describedabove and shown to prevented UV-induced lipid peroxidation by 75%. Thisis comparable to results obtained for 0.5% Vitamin E plus 0.1% Emblica.However, although the compositions were similar in ability to inhibitlipid peroxidation, by combining Vitamin E with Oxynex® ST we haveobserved an unexpected increase in protection from ROS formation acrossthe full thickness of epidermis.

These results also confirm that Oxynex® ST maintains its antioxidantcapability within skin when exposed to UVR as opposed to becoming apro-oxidant. Taken together, these results demonstrate the unexpectedbenefits of combining Vitamin E with Oxynex® ST for protection againstUV-induced ROS formation within the full thickness of epidermis.

Cosmetic Clinical Efficacy Evaluation of High SPF AntioxidantFormulation

Two topical antioxidant sunscreen formulation were generated accordingto the methods of the invention containing the sunscreen and antioxidantloads:

Ingredient SPF 70 SPF 30 Sunscreen Homosalate  15%   15% Octocrylene 10%    2% Avobenzone   3%    2% Oxybenzone   6%    5% Octisalate   5%   5% Antioxidant Diethylhexyl sringylidene 0.9% — malonate Vitamin E0.5%  0.5% Emblica — 0.01% All percentages are w/w.

In vivo SPF testing conducted according to the U.S. F.D.A. approvedtesting protocols determined that the first formulation rated as atleast SPF 70 and the second was at least SPF 30. For convenience theywill be referred to herein as SPF 70 and SPF 30. Methods of in vitro andin vivo measurement of SPF are describe, e.g., in U.S. PatentApplication Publication Nos. 20070160549 and 20080081024.

A clinical efficacy evaluation was conducted to determine the effect ofthe high SPF antioxidant formulations produced as described above onvarious indications of skin health including, skin dryness (moisture),skin texture (roughness, smoothness), elasticity (i.e., firmness orresiliency), skin tone and clarity, uniformity of pigmentation, finelines and wrinkles, erythema, photodamage, and hidden damage(subclinical pigmentation). The study is a single-blind, parallel,randomized, controlled, twelve (12) week use test with an additionalbaseline equilibration period of seven (7) days. One-hundred-nine (109)subjects were enrolled and one-hundred-five (105) continued on thestudy. Four (4) subjects were discontinued due to inability to make allregularly scheduled visits. No adverse experiences have been reported.The results below demonstrate significant improvement over baselinecondition of the skin as a result of 12 weeks of use of the testedproducts.

Qualified subjects were divided into two (2) product groups and anuntreated control group. An expert clinical evaluator graded the face ofeach subject at each visit to assess individual parameters thatcontribute to the visual and tactile properties of premature aging, aswell as to provide an overall global assessment of degree of visiblephotodamage. The clinical grading scores at baseline were used toconfirm that the subject presents with mild to moderate photo-damage andtherefore were qualified for participation.

Specific attributes were quantified using bioinstrumentation; silasticresin replicas with image analysis to also measure fine lines andwrinkles, a Nova Meter for determining moisture content, and a Dermalabsuction device to measure skin elasticity. A trained photographerphotographed each subject at baseline, 2, 4, 8 and 12 weeks using afixed angle standard and cross-polarized light Canfield clinical cameraapparatus to document appearance of a specific site on the side of theface (including crow's feet area). The photographer took full facial UVreflected photographs, at baseline and at the 12-week visit or until theproduct had washed out and no longer fluoresces (whichever was later).The expert clinical evaluator graded the UV photographs taken atbaseline and 12-weeks (±days to washout) to assess degree of subclinical“hidden” damage present and then globally assessed the amount of changecompared to baseline.

Test compositions were overwrapped to hide the identity of themanufacturer and labeled with the appropriate test article codes and usedirections. Approximately one-half the study product was delivered priorto the start of the study and the second half of the product wasdelivered prior to the mid-point of the study.

Seven to ten days prior to initiation of the treatment period, subjectsunderwent a baseline equilibration period, during which theydiscontinued the use of all facial sunscreens, skin treatment products,their current facial cleansing bar or cream, and any moisturizing facialcosmetic products; use Camay soap daily, each morning for any facialcleansing and as needed throughout the day; and refrain from use oftanning beds for the duration of the study.

Following the equilibration period, subjects were qualified bypresenting with sufficient signs of dryness and extrinsic skin aging.Following qualification, subjects were randomly assigned to one of twotest groups or to the untreated control group. For the duration of thestudy subjects assigned to both the treatment and non-treatment groupswash their faces only with the Camay soap provided. Subjects in both ofthe treatment groups applied the assigned test article to their face(and neck if desired) once daily (each morning), then reapply as needed.

Subjects recorded application times each day on a diary provided by thestudy site at each visit. Subjects in the non-treatment group recordedthe number of times that they cleanse and apply their usual moisturizer,sunscreen and color cosmetic products during the study. Diaries werecollected and redistributed in the same manner as outlined for thetreatment groups.

All subjects had clinical skin evaluations, Nova Meter, Dermalab,Silastic resin replicas, and standard light photography after 2, 4, 8and 12 weeks. Subjects have UV photos taken only at baseline and 12weeks (±days for washout) by an expert clinical evaluator who was notaware of product assignment, nor which subjects were in the treatmentgroups and which subjects were in the non-treated control group.

Evaluations were made at each visit for several indicators ofphotodamage listed below. Grading scales are outlined in each of thecategories

A. Overall Assessment of Degree of Photodamage.

Subjects were graded on a scale of 0-10 with 0 representing nophotodamage and 10 representing severe photodamage. The results were asfollows:

TABLE 9 MEAN OVERALL PHOTODAMAGE SCORE ± S.D. (% IMPROVEMENT) SPF 30 SPF70 CONTROL BASELINE 4.80 ± 0.97 4.72 ± 0.84 4.71 ± 0.79 (n = 39) (n =39) (n = 27) WEEK 2 4.41 ± 0.73* (8) 4.45 ± 0.51* (5) 4.44 ± 0.60* (6)(n = 39) (n = 39) (n = 27) WEEK 4 4.04 ± 0.62* (16) 4.15 ± 0.68* (12)4.14 ± 0.60* (12) (n = 39) (n = 39) (n = 27) WEEK 8 4.40 ± 0.70* (8)4.42 ± 0.51*(6) 4.48 ± 0.64^(T)(5) (n = 39) (n = 37) (n = 26) WEEK 123.97 ± 0.49* (17) 3.86 ± 0.38* (18) 3.95 ± 0.38* (16) (n = 39) (n = 39)(n = 27) *Significantly different than baseline value, p ≦ 0.050.^(T)Trendwise significantly different than baseline value,p-0.150-0.051.

B. Facial Dryness.

Subjects were given grades of 0-4 as follows:

-   -   0 normal skin; no signs of dryness    -   1 mild dryness; slight but definite dryness, fine scaling        present may have a powdery or ashy appearance    -   2 moderate dryness; somewhat coarser scaling, some cracking        evident as uplifted scales.    -   3 marked dryness; marked coarse scaling, cracking evident as        uplifted scales.    -   4 severe dryness; very marked; very coarse scaling; cracking        progressing to fissuring; erythema may be present.        The results were as follows:

TABLE 10 MEAN DRYNESS SCORE ± S.D. (% IMPROVEMENT) SPF 30 SPF 70 CONTROLBASELINE 0.97 ± 0.16 1.05 ± 0.22 1.07 ± 0.26 (n = 39) (n = 39) (n = 27)WEEK 2 0.20 ± 0.52* (79) 0.23 ± 0.48* (78) 0.11 ± 0.32* (90) (n = 39) (n= 39) (n = 27) WEEK 4 0.12 ± 0.40* (88) 0.15 ± 0.36* (86) 0.18 ± 0.48*(83) (n = 39) (n = 39) (n = 27) WEEK 8 0.33 ± 0.57*(66) 0.10 ± 0.31*(91)0.26 ± 0.53*(76) (n = 39) (n = 37) (n = 26) WEEK 12 0.00 ± 0.00* (100)0.00 ± 0.00* (100) 0.11 ± 0.32* (90) (n = 39) (n = 39) (n = 27)

C. Texture (Roughness/Smoothness)

Subjects were graded from a score of 0, indicating smooth, even surface,to 10 indicating a rough, coarse, uneven surface. The results were asfollows:

TABLE 11 MEAN TEXTURE SCORE ± S.D. (% IMPROVEMENT) SPF 30 SPF 70 CONTROLBASELINE 3.68 ± 0.69 3.86 ± 0.61 3.64 ± 0.63 (n = 39) (n = 39) (n = 27)WEEK 2 2.24 ± 1.00* (39) 2.56 ± 0.94* (34) 2.35 ± 1.02* (35) (n = 39) (n= 39) (n = 27) WEEK 4 2.58 ± 0.97* (30) 2.25 ± 1.17* (42) 2.30 ± 1.02*(37) (n = 39) (n = 39) (n = 27) WEEK 8 2.62 ± 0.87*(29) 2.88 ± 0.95*(25)2.70 ± 0.87*(26) (n = 39) (n = 37) (n = 26) WEEK 12 2.52 ± 0.74* (32)2.35 ± 0.73* (39) 2.64 ± 0.64* (28) (n = 39) (n = 39) (n = 27)*Significantly different than baseline value, p ≦ 0.050.

D. Elasticity/Firmness/Resiliency

Subjects were graded from a score of 0, indicating firm, resilient, tautskin, to 10 indicating skin that was loose, flaccid, no turgor. Theresults were as follows:

TABLE 12 MEAN ELASTICITY/FIRMNESS/RESILIENCY SCORE ± S.D. (%IMPROVEMENT) SPF 30 SPF 70 CONTROL BASELINE 5.16 ± 1.13 5.18 ± 0.94 5.21± 1.03 (n = 39) (n = 39) (n = 27) WEEK 2 4.24 ± 1.13* (18) 4.19 ± 1.06*(19) 4.21 ± 1.20* (19) (n = 39) (n = 39) (n = 27) WEEK 4 4.15 ± 0.97*(20) 4.00 ± 0.80* (23) 4.30 ± 1.08* (18) (n = 39) (n = 39) (n = 27) WEEK8 3.77 ± 0.94*(27) 3.81 ± 0.92*(26) 4.05 ± 1.00* (22) (n = 39) (n = 37)(n = 26) WEEK 12 3.33 ± 0.92* (36) 3.29 ± 1.03* (37) 3.67 ± 0.87* (30)(n = 39) (n = 39) (n = 27) *Significantly different than baseline value,p ≦ 0.050.

E. Lines and Wrinkles

Subjects were graded from a score of 0, indicating no lines or wrinkles,to 10 indicating coarse skin containing numerous wrinkles. The resultswere as follows:

TABLE 13 MEAN FINE LINES/WRINKLES SCORE ± S.D. (% IMPROVEMENT) SPF 30SPF 70 CONTROL BASELINE 4.12 ± 1.42 4.78 ± 1.32 4.44 ± 1.58 (n = 39) (n= 39) (n = 27) WEEK 2 3.92 ± 1.18 (5) 4.28 ± 0.72* (11) 4.37 ± 1.21 (2)(n = 39) (n = 39) (n = 27) WEEK 4 4.12 ± 0.95 (0) 4.15 ± 0.80* (13) 4.07± 1.29^(T) (8) (n = 39) (n = 39) (n = 27) WEEK 8 3.55 ± 0.90*(14) 3.75 ±0.74*(22) 3.81 ± 0.97*(14) (n = 39) (n = 37) (n = 26) WEEK 12 3.28 ±0.89* (20) 3.58 ± 0.69* (25) 3.49 ± 1.06* (21) (n = 39) (n = 39) (n =27) *Significantly different than baseline value, p ≦ 0.050.^(T)Trendwise significantly different than baseline value,p-0.150-0.051.

F. Skin Tone/Clarity

Subjects were graded from a score of 0, indicating clear, radiant,translucent skin, to 10 indicating skin that was sallow, dull and/or haduneven skin tone. The results were as follows:

TABLE 14 MEAN SKIN TONE SCORE ± S.D. (% IMPROVEMENT) SPF 30 SPF 70CONTROL BASELINE 5.57 ± 0.81 5.40 ± 0.65 5.44 ± 0.85 (n = 39) (n = 39)(n = 27) WEEK 2 5.07 ± 0.69* (9) 5.11 ± 0.60* (5) 5.33 ± 0.56 (2) (n =39) (n = 39) (n = 27) WEEK 4 4.90 ± 0.70* (12) 4.78 ± 0.77* (12) 4.84 ±0.76* (11) (n = 39) (n = 39) (n = 27) WEEK 8 4.62 ± 0.68*(17) 4.50 ±0.69*(17) 4.53 ± 0.79*(17) (n = 39) (n = 37) (n = 26) WEEK 12 4.14 ±0.75* (26) 3.92 ± 0.80* (27) 3.98 ± 0.62* (27) (n = 39) (n = 39) (n =26) *Significantly different than baseline value, p ≦ 0.050.

G. Uniformity of Pigmentation

Subjects were graded from a score of 0, indicating uniform, evenpigmentation, to 10 indicating skin that was uneven, blotchy or mottled.The results were as follows:

TABLE 15 MEAN UNIFORMITY OF PIGMENTATION SCORE ± S.D. (% IMPROVEMENT)SPF 30 SPF 70 CONTROL BASELINE 5.07 ± 0.94 4.90 ± 0.99 4.95 ± 1.09 (n =39) (n = 39) (n = 27) WEEK 2 4.80 ± 0.74^(T) (5) 4.67 ± 0.77^(T) (5)4.76 ± 0.73 (4) (n = 39) (n = 39) (n = 27) WEEK 4 4.99 ± 0.89 (2) 4.81 ±0.90 (2) 4.84 ± 0.84 (2) (n = 39) (n = 39) (n = 27) WEEK 8 4.42 ±0.66*(13) 4.38 ± 0.59*(11) 4.37 ± 0.78*(12) (n = 39) (n = 37) (n = 26)WEEK 12 4.14 ± 0.73* (18) 4.04 ± 0.78* (18) 3.97 ± 0.69* (20) (n = 39)(n = 39) (n = 27) *Significantly different than baseline value, p ≦0.050. ^(T)Trendwise significantly different than baseline value,p-0.150-0.051.

H. Erythema

Subjects were graded from a score of 0, indicating no erythema or normaltone, to 10 indicating skin that was marked, very red. The results wereas follows:

TABLE 16 MEAN NOVA METER VALUE ± S.D. (% IMPROVEMENT) SPF 30 SPF 70CONTROL BASE- 202.55 ± 87.93 203.12 ± 88.65 218.92 ± 114.92 LINE (n =39) (n = 39) (n = 27) WEEK 2 179.17 ± 64.82^(T) 180.15 ± 74.05^(T)196.22 ± 86.17 (−12) (−11) (−10) (n = 39) (n = 39) (n = 27) WEEK 4160.17 ± 42.24* 177.0 ± 72.68* 191.74 ± 77.53 (−21) (−13) (−12) (n = 39)(n = 39) (n = 27) WEEK 8 158.97 ± 39.01* 150.10 ± 38.63* 166.73 ± 57.76*(−22) (−26) (−24) (n = 39) (n = 37) (n = 26) WEEK 12 169.89 ± 45.02*182.35 ± 60.00^(T) 179.59 ± 62.21* (−16) (−10) (−18) (n = 39) (n = 39)(n = 27) *Significantly different than baseline value, p ≦ 0.050.^(T)Trendwise significantly different than baseline value, p =0.150-0.051.

Facial skin condition was measured on all subjects using Dermalab,Novameter and replica image analysis at baseline and weeks 2, 4, 8 and12 as follows.

Elasticity was measured on one side of the face (same location at eachvisit) on all subjects using the Dermalab (Cortex Technology, Denmark),which applies a negative pressure to the skin surface and calculates theheight to which the skin can be drawn up and the rate at which itreturns to equilibrium thus providing a measurement of elasticity.Dermalab measurements took place on the opposite side of the face asimage analysis replicas.

Moisturization was measured on one side of the face (same location ateach visit) to document hydration levels of the skin surface. Therelative degree of skin hydration is assessed using the Dermal PhaseMeter 9003 (NOVA meter). Measurements are made by applying analternating voltage to the skin with a closely spaced pair of electrodesand measuring the impedance. Changes in water content change theimpedance of the capacitive circuit. The first two consecutive readingswithin 10% were recorded. The same side of the face is measured at eachvisit. The test room temperature and humidity will be recorded duringeach set of readings.

Contour (surface textural) analysis provides a method for quantifyingskin augmentation, the cosmetic action of reducing lines and wrinkles.For this procedure, skin replicas made of the crow's feet area wereanalyzed for contour and surface texture using image analysis. Skinreplicas of the crow's feet area were prepared using silastic resinimpression materials (Cuderm). Silastic resin is a rapidly curing liquidapplied using 1 cm diameter replica rings which stay intact afterapplication and removal.

Facial skin condition was documented for all subjects using standard andcross polarized light photography at baseline and weeks 2, 4, 8 and 12.Hidden damage accumulated below the skin surface was evaluated by expertgraders, based on UV photos taken at baseline and 12-weeks.

Digital photographs using both visible and cross-polarized light aretaken of all subjects at all visits. Subjects' faces are positioned inthe Canfield stereotactic repositioning apparatus and photographs aretaken using the Canfield Clinical Systems camera and flash system. Thecamera used was a Nikon D80 SLR 35 mm model with a 60 mm macro Nikkorlens and a modified SB-23 flash head. The camera is set in Aperturepriority automatic at f I6. For each subject at each time interval, aslate was photographed at 1:6 magnification identifying the subject andtime interval. A frontal photo was taken at 1:6 magnification and twolateral 45° angle photos of each side of the face is taken at 1:4 and1:3 magnification using standard lighting and repeated usingcross-polarized light.

Subjects placed their heads in the Canfield stereotactic repositioningdevice and have photographs taken using the Canfield Clinical Systemscamera system. The camera used was a Nikon 6006 SLR 35 mm model. Foreach subject at each time interval, a slate was photographed at 1:6magnification identifying the subject and time interval. A frontal photowas taken at 1:6 magnification and one lateral 45° angle photo of eachside of the face was taken at 1:6 magnification. Subsequently, twofrontal UV-light (UV reflected) photographs were taken at 1:6magnification employing a Kodak 18A filter over the lens, a Sunpak MS4000 Monolight and T-Max 400 black and white print film. Exposures weretaken at f8 and 1/250 sec. shutter speed.

An expert evaluator graded full facial photos individually foruniformity of pigmentation at each time-point (baseline and 12-week)grading on a scale of 0 (uniform/even) to 10 (uneven, blotchy mottled).The expert evaluator also conducted a comparative assessment of 12-weekphotos vs. baseline for each individual subject. Grading scales are from−4 to +4 as follows:

−4 extreme increase in hyper pigmentation

−3 moderate increase in hyper pigmentation

−2 mild increase in hyper pigmentation

−1 barely perceptible increase (worsening) in hyper pigmentation

0 no difference between baseline and 12-week

1 barely perceptible decrease (improvement) in hyper pigmentation

2 mild in hyper pigmentation

3 moderate decrease in hyper pigmentation

4 extreme decrease in hyper pigmentation

Example Formulations

Example sunscreen formulations are prepared according to the methodsdescribed herein with the following ingredients:

TABLE 17 Ingredient Amount, % w/w Purified Water 45.0-90.0 Homosalate 5.0-15.0 Octocrylene  2.0-10 Oxybenzone  0.5-6.0 Octisalate  5.0Avobenzone  1.0-3.0 Prolipid 141  2.0-7.0 Butylene Glycol  2.0-7.0Microcrystalline Cellulose/  0.2-5.0 Carboxymethylcellulose   BenzylAlcohol  0.5-2.0 Vitamin E 0.01-3.0 Diethylhexyl syrigylidene malonte0.01-6.0 Phylanthus Emblica fruit extract 0.01-1.0 Green Tea Extract0.01-1.0 Disodium Lauriminodiproprionate  0.3-3.0 Tocopheryl PhosphatesChlorphenesin  .10-0.20 Butylated PVP  .05-.50 Disodium EDTA 0.01-.20Sodium Ascorbyl Phosphate  .01-1.0 Vitamin A Palmitate  .01-1.0

What is claimed is:
 1. An ex vivo method for screening compounds for antioxidant behavior in a composition to be topically applied to skin, wherein the screening method comprises determining the compound's ability both to inhibit ultraviolet radiation-induced skin lipid hydroperoxide formation and to inhibit ultraviolet radiation-induced reactive oxygen species formation in the epidermis; wherein determining inhibition of ultraviolet radiation-induced reactive oxygen species formation in the epidermis comprises imaging skin tissue using two-photon fluorescence intensity imaging; and wherein determining inhibition of UVR-induced skin lipid hydroperoxide formation comprises the steps of applying to distinct areas of skin of a subject an antioxidant-containing composition and a placebo composition to produce an antioxidant skin site containing antioxidant and skin lipids and a placebo skin site containing placebo and skin lipids; applying a strip to the antioxidant skin site and the placebo skin site to produce an antioxidant strip sample containing antioxidant and skin lipids and a placebo strip sample containing placebo and skin lipids; removing said strip samples from the skin and exposing said strip samples to UVR to form a UVR-induced antioxidant/lipid reaction product on the antioxidant strip sample and a UVR-induced placebo/lipid reaction product on the placebo strip sample; separately contacting the antioxidant strip sample and the placebo strip sample with solvent to prepare a first extract containing UVR-induced antioxidant/lipid reaction product and a second extract containing UVR-induced placebo/lipid reaction product; assaying said first and second extracts for lipid hydroperoxide content for each extract; and comparing the lipid hydroperoxide content of the first extract to the lipid hydroperoxide content of the second extract.
 2. The method of claim 1, wherein determining inhibition of UVR-induced skin lipid hydroperoxide formation comprises the further steps of applying placebo to two distinct sites on the skin of the subject; producing strip samples from each site; subjecting strip samples from only one of the two placebo sites to UVR to produce a subset of 